Analysis of electrophoretic bands in a substrate

ABSTRACT

A method and system to enhance analysis of electrophoretic bands by overlaying only the pixels of interest. The overlaid pixels are superimposed as a layer above, i.e., in the foreground of, the overlaid image, i.e., in the background. A user employs the superimposed pixels for molecular weight determination and is still able to generate densitometry analysis of the remaining pixels in the overlaid image.

This application claims priority to U.S. application Ser. No. 13/786,976filed Mar. 6, 2013 now U.S. Pat. No. 9,230,185 and U.S. Application Ser.No. 61/617,819 filed Mar. 30, 2012, each of which is expresslyincorporated by reference herein in its entirety.

A method and system to visualize and analyze target objects from twodifferent images of the same substrate acquired by a charge coupleddevice (CCD) imager. The method and system improved band detection bysuperimposing molecular weight markers, detected with a firstvisualizing means, over analyte bands, detected with a secondvisualizing means.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. A Petition under 37 C.F.R. §1.84 requesting acceptance of thecolor drawing is being filed separately. Copies of this patent or patentapplication publication with color drawing(s) will be provided by theOffice upon request and payment of the necessary fee.

FIG. 1 diagrams a general purpose computer system suitable for operatingthe inventive method and system.

FIG. 2 is an exemplary flowchart showing one embodiment of the methodand system.

FIG. 3 is digital image of a Western blot with bands captured by whitelight illumination.

FIG. 4 is a digital image of a Western blot with bands captured bychemiluminescence.

FIG. 5 is a digital image of a layered composite image where theleftmost lane is taken from FIG. 3, and the other lanes are taken fromFIG. 4.

FIG. 6 is a digital image similar to FIG. 5 with horizontal lines oneach band from FIG. 3.

FIG. 7 is digital image of a Western blot with bands captured by whitelight illumination.

FIG. 8 is digital image of a Western blot assay with bands captured bychemiluminescence.

FIG. 9 is digital image similar to FIG. 7 with lanes defined over theimage.

FIG. 10 is digital image similar to FIG. 8 with lanes defined over theimage.

FIG. 11 is a digital image similar to FIG. 9 with a user-identified laneto use as the visible molecular weight standard.

FIG. 12 is a digital image similar to FIG. 10 where the lane marked inFIG. 11 as the visible molecular weight standard is overplayed in thecorrect lane of the chemiluminescently visualized image of the samplesubstrate.

FIG. 13 is a sample analysis report.

FIGS. 14A-I show steps in a sample procedure.

Life science researchers routinely image stained gel electrophoresissamples and Western blots for data analysis. To perform electrophoreticseparation of complex mixtures, several samples containing thesemixtures are applied to the electrophoresis gel in separate locations.When an electrical current is applied, the individual samples migratevertically down the gel within their prescribed vertical lane or track,and generate an invisible lane on the gel. The complex mixture is thenseparated by size, i.e., molecular weight, in the gel matrix. Thelarger, higher molecular weight molecules remain relatively nearer thetop of the gel or membrane. The smaller, lower molecular weightmolecules migrate toward the bottom of the gel or membrane. Eachindividual segregation is then identified as a band. The gel can then bestained for total sample visualization, or transferred to a membrane forvisualization of a specific target of interest by Western blotting. Theresearcher then images the gel or blot, collectively termed a substrate,to analyze the target(s) of interest for amount, relative or absolute,purity, and molecular weight. Such analysis requires detection andidentification of the lanes and bands in the image.

Two images of the same substrate are typically required using differentvisualization means, such as white light illumination, fluorescence, orchemiluminescence. With Western blots, objects in one image, such asprotein molecular weight markers located on one area of the substrate,are stained with a visible dye; and objects from the other image, suchas biological samples located on other areas of the substrate, aredetected by antibody-based chemiluminescent or fluorescent techniques.The stained molecular weight markers located in one or more lanes on theblot are visualized by white light illumination and the biologicalsamples located in other lanes on the blot are visualized usingchemiluminescence. Thus, two corresponding images of the same substrateare captured with the exact same view of the substrate, but withdifferent visualization methods.

Molecular weight size markers typically are a set of molecules of knownsize, i.e., molecular weights. If such a marker set was applied to onelane in a gel and separated by electrophoresis, parallel to the unknownsamples, the vertical positions of the bands observed in the molecularweight marker set can be compared to the vertical positions of the bandsin the unknown sample in order to determine the molecular weight of themolecules in the unknown sample. The relative amount of each band iscalculated by comparing its signal intensity to that of other bands. Theabsolute amount of a band is calculated by comparing signal intensity tobands of known quantity.

Existing image analysis technology allows overlaying two or morecomplete images to generate an overall composite image, facilitatingmolecular weight determination by comparing vertical positions betweenthe two images. However, analysis of relative areas must be performed ona single image at a time. Commercial image analysis software permitscreation of a composite image by adding pixel intensities at eachcorresponding location on the images, but this prevents accurateanalysis because the pixels in the overlaid image will have the pixelvalues from the superimposed image included in the analysis.

Publicly available ImageJ software can create a composite image or imagestack from images acquired in different color channels or multiplegrayscale images of the same sample, but this composite image cannot beanalyzed. ProteinSimple's AlphaView software can overlay images tocreate a RGB image, but again the resulting composite image cannot beanalyzed. Syngene's GeneSnap software can acquire images of the samesubstrate by different detection methods and then create a compositeimage from two or three original images. The composite image can then beanalyzed by Syngene's GeneSnap or GeneTools Software functions. However,since the created composite image is achieved by pixel addition, anysubsequent band analysis is compromised. The software user manual statesthat such images “do not satisfy the conditions required for GoodLaboratory Practice” and that this is noted in the image's captureproperties. Hence these composite images should not be used fordensitometry analysis and are only appropriate for molecular weightdetermination of the samples.

The inventive system allows a user to perform the necessary molecularweight analyses from data shared between the two images, while retainingthe ability to evaluate the accurate, undistorted densitometry data fromone of the images at a time.

In one embodiment, the method and system overlays only the pixels ofinterest, where the superimposed pixels exist as a layer above, i.e., inthe foreground, the overlaid image, i.e., the background. The user isthus able to employ the superimposed pixels for other types of analysis,e.g., molecular weight determination, and is still able to generatedensitometry analysis of the remaining pixels in the overlaid image.

In one embodiment, the method and system overlays selected pixels fromone image onto the corresponding location of the other image. This pixeloverlay allows objects from the two different images to be visualized atthe same time in a resulting composite image. It allows objects from thetwo images to be analyzed independently on the composite image, andallows the user to interact with, e.g., draw and adjust a lane frame orother region of interest) both images simultaneously.

The two images of the same substrate are typically acquired usingdifferent visualization means, such as white light illumination,fluorescence, or chemiluminescence.

In one embodiment of the system, the computer displays both images tothe user simultaneously during the process used to create the area ofpixel overly. In this embodiment, the software shows both images to theuser. As the user draws the lane frame on the first image, that samelane frame is shown in the corresponding location on the second image.The first and second images are thus “linked”, so that the lane frame isexactly as the user desires before the user indicates to the softwarewhich lane to overlay. This embodiment minimizes or avoids the need forthe user to adjust the lane frame after the fact if it were not as theuser desired.

The method and system is used for life science applications. Proteinmolecular weight markers electrophoretically separated are located inone area of a substrate, and are typically stained with a visible dye.Test or analyte samples electrophoretically separated are located inother areas of a substrate and are typically detected by antibody-basedchemiluminescent or fluorescent techniques. In this embodiment, theseparated stained molecular weight markers are typically visualized bywhite light illumination; the separated test or analyte samples aretypically visualized by fluorescence or chemiluminescence. Thus, twocorresponding images of the same substrate are captured with the exactsame view of the substrate but with different visualization methods.

In use, the user-defined pixels from the first image are then overlaidby the software onto the corresponding pixels from the second image,generating a new composite image. In the composite image, thesuperimposed pixels exist as a layer above, i.e., in the foreground of,the overlaid image, i.e., in the background. This composite imageenables visualization and analysis of the superimposed objectsindependently of the objects on the overlaid image. For example, thesuperimposed pixels containing the molecular weight marker lane on ablot can be used to determine the relative position of the markers onthe blot and this information can be used to estimate the molecularweights of protein bands from biological samples visualized bychemiluminescence in other lanes in the overlaid image. The proteinbands in other lanes on the overlaid image can then be analyzed usingdensitometry to determine the relative abundance of these proteins. Theuser utilizes the superimposed pixels for one type of analysis, e.g.,molecular weight determination, and is still able to generatedensitometry analysis of the remaining pixels in the overlaid image.This process is enabled by a CCD imager programmed to automaticallyacquire a visible image prior to acquiring a chemiluminescence orfluorescent image. Thus both images are taken of the same substrate withidentical substrate location and orientation. This automatic visibleimage acquisition feature can be performed without any user input andallows subsequent overlay and band analysis as described above. Thecombination of automatic visible image acquisition in the hardware andthe overlay analysis in the software provides user convenience andutility.

In one embodiment, the inventive method uses an algorithm to enhanceanalysis and enable automatic identification of bands on a gel or blotby removing noise from the identified objects or bands. The method usesoptimized threshold values, where a user-defined portion of one image issuperimposed on another image in such a way that the pixel values fromthe two images are maintained, the composite image with the user-definedportion of the superimposed image is simultaneously displayed on top ofthe overlaid image, image analysis is performed on the composite image,i.e., molecular weight determination is performed on the superimposedimage, which can be applied to the overlaid image lanes, and accurate,undistorted densitometry can be performed on the underlying imagewithout the additive pixel values from the superimposed image.

In one embodiment, the method and system are used with results from ablotting procedure. In a blotting procedure, proteins and/or nucleicacids (deoxyribonucleic acids (DNA) and/or ribonucleic acids (RNA)) in abiological sample are first electrophoretically separated from eachother, typically based upon their size, on a substrate or medium such asa sodium dodecyl sulfate-polyacrylamide gel. Next, a detectable probethat binds to a specific protein(s) or nucleic acid(s), or type ofprotein(s) or nucleic acid(s), is contacted with the substrate ormedium. The probe may be, e.g., an antibody that specifically binds to aprotein. The probe may be labeled with a compound that renders itdetectable, e.g., a chemiluminescent compound detected by itschemiluminescence. Other ways to detect a probe are known to a personskilled in the art and include, but are not limited to, radioisotopelabeling of the probe and detection by scintillation counting.

Protein(s), DNAs, and/or RNAs of interest are separated and thendetected in blotting procedures (Western, Southern, and Northern blots,respectively). The results of the blotting procedure appear as a ladderwhere rungs of the ladder are the separated proteins or nucleic acids.If the labeled probe is bound, these rungs are visualized when theappropriate detection means are used (e.g., chemiluminescent probes aredetectable upon chemiluminescent detection). The location(s) of theprotein(s) or nucleic acid step(s) in the analyzed sample is compared tothe location of the protein(s) or nucleic acid(s) steps in a controlsample containing qualitatively and/or quantitatively known protein(s)and/or nucleic acid(s). Label detection permits identification of thepresence, size, and/or concentration of the protein(s) or nucleicacid(s) based on the location and/or intensity of the signal. Thepattern produced by this procedure is captured and recorded using animage capturing device such as autoradiography film, charge-coupleddevice (CCD) camera, scanner, phosphor imager, or other capture deviceknown to a person skilled in the art. A durable copy of the image,termed a blot, records the results, permitting data comparison,memorialization, etc.

CCD cameras are a method of choice for image capture of scientificresearch results such as those from SDS-PAGE gel staining and blottingmethods (Western, Northern and Southern). A common detection methodutilized for identifying a target of interest in a blotting assay ischemiluminescence. However the most common control samples containingknown protein(s), called molecular weight markers, are visibly labeledthus rendering them unable to be viewed by chemiluminescence.Historically, the signal generated from chemiluminescent blots has beencaptured using X-ray or autoradiography film. Results of the molecularweight markers are hand-drawn onto the exposed film with permanent inkand are only able to be used for qualitative approximation of sizeand/or amount of the protein(s) or nucleic acid(s) based on the locationand/or intensity of the signal. Advances in digital imaging technologyhave made it possible to obtain results using instrumentation thatcaptures a digital image, versus using conventional film. New imagecapture devices such as CCD camera systems improve image qualitycompared to film.

To enhance a researcher's ability to obtain a chemiluminescent signalfrom a blot and a visible signal from a stained molecular weight marker,the inventive methods and systems automatically acquire a visible imageof every blot imaged in the chemiluminescence acquisition mode. Thevisible image is taken with white light illumination. Thechemiluminescent image is acquired without illumination. Thus the imagesare taken of the same sample with identical sample locations. Theseimages are then processed by the inventive overlay method to identifythe presence, size, and/or concentration of the protein(s) and/ornucleic acid(s). Images from other instrumentation can be used only ifthe user manually acquires the two images of the same gel usingdifferent detection methods (i.e., white illumination andchemiluminescence or fluorescence) with identical sample location. Incontrast, the inventive methods and systems automatically perform thisfunction.

In one embodiment, the inventive method and system combines informationfrom two images of the same blot or sample, shown in the followingsteps: open image, detect objects on image using object detectionalgorithm, retain the target objects and triage noise objects usingthreshold, determine lanes by tracking the vertically aligned objectsfrom the top line of image, display lane and band perimeters. In anotherembodiment, the steps are: open images, perform image adjustments,create and adjust lane frame, identify image for superimposition, locatelane to be superimposed, identity molecular weight marker, andregression method to be used, detect objects on image using objectdetection algorithm, retain the target objects and triage noise objectsusing threshold, determine lanes by tracking the vertically alignedobjects from the top line of image, software calculates analysis resultsthat are displayed in an analysis table. Invention implementation in thesoftware program includes placement and adjustment of the lane frameonly while viewing the overlaid image, or while viewing both images (thelane frame can be viewed and adjusted simultaneously on both images asthe information is linked).

The inventive methods and systems improved band detection bysuperimposing molecular weight markers, detected with a firstvisualizing means, over analyte bands, detected with a secondvisualizing means. Embodiments include a method, data processing systemand/or computer program product. Thus, one embodiment is entirelyhardware with logic embedded in circuitry, one embodiment is entirelysoftware with logic operating on a general purpose computer to performthe method and operate the system, and/or one embodiment combinessoftware and hardware aspects. One embodiment takes the form of acomputer program product on a computer-readable storage medium havingcomputer readable program code means embodied in the medium. Anysuitable computer readable medium may be used including hard disks,CD-ROMs, optical storage devices, static or nonvolatile memorycircuitry, or magnetic storage devices and the like. The executableprogram may be available for download from a website.

FIG. 1 shows an exemplary computer system 100 that can be used toimplement the method and system. The computer system can be a laptop,desktop, server, handheld device (e.g., personal digital assistant(PDA), smartphone), programmable consumer electronics or programmableindustrial electronics.

As illustrated the computer system includes a processor 102 that can beany various available microprocessors. For example, the processor can beimplemented as dual microprocessors, multi-core and other multiprocessorarchitectures. The computer system includes memory 104 that can includevolatile memory, nonvolatile memory or both. Nonvolatile memory caninclude read only memory (ROM) for storage of basic routines fortransfer of information, such as during computer boot or start-up.Volatile memory can include random access memory (RAM). The computersystem can include storage media 106 including, but not limited to,magnetic or optical disk drives, flash memory, and memory sticks. Thecomputer system incorporates one or more interfaces, including ports 108(e.g., serial, parallel, PCMCIA, USB, FireWire) or interface cards 110(e.g., sound, video, network, etc.) or the like. In embodiments, aninterface supports wired or wireless communications. Input is receivedfrom any number of input devices 112 (e.g., keyboard, mouse, joystick,microphone, trackball, stylus, touch screen, scanner, camera, satellitedish, another computer system and the like). The computer system outputsdata through an output device 114, such as a display (e.g. CRT, LCD,plasma), speakers, printer, another computer or any other suitableoutput device.

The following description references flowchart illustrations of methods,apparatus (systems) and computer program products. It will be understoodthat each block of the flowchart illustrations, and combinations ofblocks in the flowchart illustrations, can be implemented by computerprogram instructions. These computer program instructions may be loadedonto a computer or other programmable data processing apparatus orotherwise encoded into a logic device to produce a machine, such thatthe instructions which execute on the computer or other programmabledata processing apparatus create means for implementing the functionsspecified in the flowchart block or blocks. These computer programinstructions may also be stored in a computer readable memory that candirect a computer or other programmable data processing apparatus tofunction in a particular manner, such that the instructions stored inthe computer readable memory produce an article of manufacture includinginstruction means that implement the function specified in the flowchartblock or blocks. The computer program instruction may also be loadedonto a computer or other programmable data processing apparatus to causea series of operational steps to be performed on the computer or otherprogrammable apparatus to produce a computer implemented process suchthat the instructions which execute on the computer or otherprogrammable apparatus provide steps for implementing the functionsspecified in the flowchart block or blocks.

As a person of ordinary skill in the art appreciates, specificfunctional blocks presented in relation to the inventive methods andsystems are programmable as separate modules or functional blocks ofcode. These modules are capable of being stored in a one- ormultiple-computer storage media in a distributed manner. In oneembodiment, these modules are executed to perform the inventive methodand system in whole or in part on a single computer. In one embodiment,these modules are executed to perform the inventive methods and systemson multiple computers that cooperatively execute the modules. In oneembodiment, the programs are executed in a virtual environment, wherephysical hardware operates an abstract layer upon which the inventivemethods and systems are executed in whole or in part across one or morephysical hardware platforms. In one embodiment, with reference to FIGS.2, 3, and 4, the inventive methods and systems provides enhancedanalysis, through a series of steps, of bands that have beenelectrophoretically separated in a substrate. In the first step, twoimages of the substrate are received 210. The first image 300 iscomposed of bands 310, 330 captured by a first visualizing means such aswhite light illumination, and the vertical position of each of theelectrophoretic bands 310, 330 is associated with a predefined molecularweight. The second image 400 is composed of bands 420 captured by asecond visualizing means such as lack of illumination, e.g., noillumination for chemiluminescence, ultraviolet illumination, LED whiteillumination or other white epi-illumination modules, or LED colorillumination; with ultraviolet, color, or white illumination optionallyin conjunction with an emission filter. Next, a layered composite imageis created 220 from the first image 300 and second image 400. Finally,the layered composite image is analyzed 230 to determine informationabout the samples in the substrate.

In one embodiment, the creating step 220 includes identifying 221corresponding pixels from a subset of pixels from the first layer thatcorrespond to a subset of pixels from the second layer; and thenorienting 222 the first layer with the second layer based at least on analignment of the corresponding pixels, resulting in the creation of thelayered composite image.

In one embodiment, the orienting step 222 includes mapping the positionsof a first set of pixels to the positions of a second set of pixels,where the first set of pixels substantially define the first layer andthe second set of pixels substantially define the second layer. Mappingis performed in accord with the positions of the corresponding pixelsresulting in a common coordinate system between the first layer and thesecond layer.

In one embodiment, noise is reduced on a single layer relative toitself, or is reduced on both layers but each layer is relative toitself.

In one embodiment, the layered composite image is visually displayed 240to a user.

In one embodiment, the analyzing step 230 compares information betweenthe first image 300 and the second image 400 and includes determiningmolecular weight 232 by measuring 234 the vertical position of a secondband 420 from the second layer; then matching 236 the vertical positionof the second band 420 to a substantially equivalent vertical positionof a first band 330 on the vertical weight scale from the first layer,and identifying a molecular weight 232 associated with the second band420.

In one embodiment, the analyzing step 230 includes identifying amolecule, e.g., a protein, associated with the molecular weight, andassociating the second band with the identified molecule.

In one embodiment, the analyzing step includes repeating the previousanalyzing steps 230 for those second bands 420 not associated with amolecule. The repeating step can continue until sufficient second bands420 have been associated with molecules that their source is establishedwith reasonable certainty, as known by one skilled in the art.

In one embodiment, the analyzing step 230 calculates information withinthe first image 300, including measuring an individual area and signalintensity 233, and purity of each of the first bands 310, 330, andreporting such densitometry analysis parameters 235 in a table. Multipledensitometry parameters are reported, including volume (signalintensity), area, density (intensity/area), median signal intensityvalue, background corrected intensity and density values (calculated bydefault and optionally user-defined background methods), and percentpurity calculated by band (single band signal intensity divided by thetotal intensity of all bands within the lane), and by lane (single bandsignal intensity divided by the total intensity within the lane).Optionally, relative and absolute amounts 231 can also be analyzed. Therelative amount of each band is calculated by comparing its signalintensity to that of other bands. The absolute amount of each band iscalculated by comparing signal intensity to bands of known quantity.

In one embodiment, the analyzing step 230 calculates information withinthe second image 400, including measuring an individual area and signalintensity 233, and purity for each of the second bands 420, andreporting such densitometry analysis parameters 235 in a table. Multipledensitometry parameters are reported, including volume (signalintensity), area, density (intensity/area), median signal intensityvalue, background corrected intensity and density values (calculated bydefault and optionally user-defined background methods), and percentpurity calculated by band (single band signal intensity divided by thetotal intensity of all bands within the lane), and by lane (single bandsignal intensity divided by the total intensity within the lane).Optionally, relative and absolute amounts 231 can also be analyzed. Therelative amount of each band is calculated by comparing its signalintensity to that of other bands. The absolute amount of each band iscalculated by comparing signal intensity to bands of known quantity.

In one embodiment, an imaging device is controlled to capture the firstimage 300 by the first visualizing means, then to capture the secondimage 400 by a second visualizing means.

In use, a user opens the two images (for simplicity, the image with noillumination e.g., chemiluminescent is noted as Image #1, and the imagewith white light illumination is noted as Image #2) for overlay andadjusts the contrast on Image #1. The user then creates and places alane frame on Image #1 and adjusts the lanes to fit the imaged sample,e.g., modify lane frame height, width, and placement; apply lane skew ifnecessary, etc. The user then indicates to the software the identity ofImage #2, the location of the lane to be replaced on Image #1,optionally the identity of the molecular weight marker which can beobtained from a dropdown menu of preloaded markers or imported, theregression method to be applied for molecular weight determination, andperforms automatic band identification which uses an optimized algorithmfor object detection and identification. The overlaid lane is depictedon the image once the user establishes the location of the lane to beoverlaid and the identity of Image #2.

One embodiment uses the inventive methods and systems with the MYECL™Imager (Thermo Fisher Scientific) implemented in the automatic lane andband identification in the myImageAnalysis™ Software (PierceBiotechnologies, Inc., Rockford Ill.). Software for the method is bothseparately available and as a component of the MYECL™ Imager, and iscapable of preparing an analysis report, a sample of which is shown inFIG. 13, with the overlaid image and analysis table, or the overlaidimage alone, and exporting this report to MicroSoft Word, MicroSoftPowerPoint, Adobe, etc. The procedure may include the following steps,with illustrations as shown in FIGS. 14A-I

Open chemiluminescent and visible images (ChemiS and ChemiV image filesfrom myECL® Imager) (FIG. 14A).

Select the Image tab and adjust chemiluminescent image contrast with theWhite Level slider bar or Auto Adjust button until bands are visible(FIG. 14B).

In the Lanes/Bands subtab, manually add the appropriate number of lanesto the chemiluminescent image, including the lane which contains thecolorimetric molecular weight marker (FIG. 14C).

Resize the Lane Frame to fit the imaged blot; adjust lane placement,width and/or skew as necessary (FIG. 14D).

In the Molecular Weight subtab, select the visible image file name inthe MWM Overlay drop-down menu (FIG. 14E).

In the Marker Lanes box, select the lane containing the molecular weightmarker (FIG. 14F).

In the Markers drop-down menu, select the appropriate molecular weightmarker. In the Regression drop-down menu, select the appropriateregression method.

Select Apply MW Markers (FIG. 14G).

Check the Find Bands box in the Auto-Analyze module and select Run (FIG.14H).

Adjust the sensitivity level in the Sensitivity tool to identify allmarker bands and bands of interest. Select OK. Note: The sensitivitylevel may need to be 100% for all marker bands to be identified. Removeundesired bands in the chemiluminescent image with the Delete Bandsbutton in the Lanes/Bands subtab.

The result is a display of the colorimetric molecular weight markerimage within the chemiluminescent image. Molecular weight determinationis performed for the located bands and results are displayed in theAnalysis Table. Accurate densitometry analysis can only be performed onthe chemiluminescent image. Pixel intensities in the marker lane on theAnalysis Table are from the visible image; all other pixel intensitiesin the Analysis Table are from the chemiluminescent image (FIG. 14I).

Imager automatically acquiring a visible image of a blot when theinstrument is in chemiluminescent acquisition mode.

The embodiments described in the specification are only specificembodiments of the inventors who are skilled in the art and are notlimiting. Therefore, various changes, modifications, or alterations tothose embodiments may be made without departing from the spirit of theinvention or the scope of the following claims.

What is claimed is:
 1. A method for improving analysis ofelectrophoretic bands in a substrate, the method comprising receiving afirst image of the substrate and a second image of the substrate, thefirst image comprising a plurality of first electrophoretic bandscaptured by a first visualizing means, and the second image comprising aplurality of second electrophoretic bands captured by a secondvisualizing means, where the vertical position of each of the pluralityof first electrophoretic bands is associated with a predefined molecularweight such that the first image represents a vertical weight scale;creating a layered composite image where the first image defines a firstlayer of the layered composite image and the second image defines asecond layer of said layered composite image; and analyzing the layeredcomposite image by a molecular weight analysis and a densitometryanalysis, where the molecular weight analysis includes performing amolecular weight determination by comparing information between thefirst image and the second image as it exists in the layered compositeimage, and where the densitometry analysis includes performing adensitometry determination by comparing information only within thesecond layer.
 2. The method of claim 1 where the creating step furthercomprises identifying a plurality of corresponding pixels defined from asubset of pixels from the first layer that correspond to a subset ofpixels from the second layer; and orienting the first layer with thesecond layer based at least on an alignment of the plurality ofcorresponding pixels, resulting in creating the layered composite image.3. The method of claim 2 where the orienting step further comprisesmapping the positions of a first set of pixels to the positions of asecond set of pixels, where the first set of pixels substantiallydefines the first layer, and the second set of pixels substantiallydefines said second layer, where mapping is performed in accordance withthe positions of the plurality of corresponding pixels resulting in acommon coordinate system between the first layer and the second layer.4. The method of claim 1 where the molecular weight determinationcomprises determining the vertical position of a second electrophoreticband from the second layer; matching the vertical position of the secondelectrophoretic band to a substantially equivalent vertical position onthe vertical weight scale from the first layer; and identifying amolecular weight associated with the second electrophoretic band.
 5. Themethod of claim 4 where the analyzing step further comprises identifyinga molecule associated with the molecular weight; and associating thesecond electrophoretic band with the identified molecule.
 6. The methodof claim 5 where the analyzing step further comprises repeating theprevious analyzing steps for those second electrophoretic bands notassociated with a molecule.
 7. The method of claim 6 wherein therepeating step continues until a sufficient number of secondelectrophoretic bands have been associated with molecules to establish asource of the second electrophoretic bands.
 8. The method of claim 1where the analyzing step compares information within the first image andcomprises measuring an individual area for each of the plurality offirst electrophoretic bands; measuring signal intensity for each of theplurality of first bands; and calculating a percent purity for eachband.
 9. The method of claim 1 where the densitometry determinationcomprises measuring an individual area for each of the plurality ofsecond electrophoretic bands; measuring signal intensity for each of theplurality of second bands; and calculating a percent purity for eachband.
 10. The method of claim 1 further comprising controlling animaging device to capture the said first image by a first visualizingmeans, then to capture the second image by a second visualizing means.11. A non-volatile computer readable storage medium having data storedtherein representing software executable by a computer, the softwareincluding instructions to provide improved analysis of electrophoreticbands in a substrate, the storage medium comprising instructions forreceiving a first image of the substrate and a second image of thesubstrate, where the first image comprises a plurality of firstelectrophoretic bands captured by a first visualizing means, and thesecond image comprises a plurality of second electrophoretic bandscaptured by a second visualizing means, where the vertical position ofeach of the plurality of first electrophoretic bands is associated witha predefined molecular weight such that said first image represents avertical weight scale; instructions for creating a layered compositeimage, where the first image defines a first layer of the layeredcomposite image and the second image defines a second layer of thelayered composite image; and instructions for analyzing the layeredcomposite image, where the instructions for analyzing comprise a firstset of instructions and a second set of instructions, where the firstset of instructions include performing a molecular weight determinationby comparing information between the first image and the second image asit exists in the layered composite image, and where the second set ofinstructions include performing a densitometry determination bycomparing information only within the second layer.
 12. The non-volatilecomputer readable storage medium of claim 11 where the instructions forcreating a layered composite image further comprise instructions foridentifying a plurality of corresponding pixels defined from a subset ofpixels from the first layer that correspond to a subset of pixels fromthe second layer; and instructions for orienting the first layer withthe second layer based at least on an alignment of the plurality ofcorresponding pixels, resulting in the creation of the layered compositeimage.
 13. The non-volatile computer readable storage medium of claim 12where the instructions for orienting the first layer with the secondlayer further comprise instructions for mapping the positions of a firstset of pixels to the positions of a second set of pixels, where thefirst set of pixels substantially define the first layer, and the secondset of pixels substantially define the second layer, where the mappingis performed in accord with the positions of the plurality ofcorresponding pixels, resulting in a common coordinate system betweenthe first layer and the second layer.
 14. The non-volatile computerreadable storage medium of claim 11 where the first set of instructionsfor analyzing comprise instructions for determining the verticalposition of a second electrophoretic band from the second layer;instructions for matching the vertical position of the secondelectrophoretic band to a substantially equivalent vertical position onthe vertical weight scale from the first layer; and instructions foridentifying a molecular weight associated with the secondelectrophoretic band.
 15. The non-volatile computer readable storagemedium of claim 11 where the instructions for analyzing are directed tocomparing information within the first image and comprises instructionsfor measuring an individual area for each of the plurality of firstelectrophoretic bands; instructions for adding each of the individualareas to acquire a total area; and instructions for calculating apercent of total weight for each of the plurality of firstelectrophoretic bands, where each calculation takes the individual areafor each band and divides that value by the total area for all bands.16. The non-volatile computer readable storage medium of claim 11 wherethe second set of instructions for analyzing comprise instructions formeasuring an individual area for each of the plurality of secondelectrophoretic bands; instructions for adding each of the individualareas to acquire a total area; and instructions for calculating apercent of total weight for each of the plurality of secondelectrophoretic bands, where each calculation takes the individual areafor each band and divides that value by the total area for all bands.17. The non-volatile computer readable storage medium of claim 11further comprising instructions for controlling an imaging device tocapture the first image by the first visualizing means, and instructionsto capture the second image by a second visualizing means.
 18. A systemfor improving analysis of electrophoretic bands in a substrate, thesystem comprising an imaging device adapted to capture a plurality ofdigital images including a first image of the substrate and a secondimage of the substrate, where the imaging device captures the firstimage by a first visualizing means and immediately thereafterautomatically captures the second image by a second visualizing means,where the first image comprises a plurality of first electrophoreticbands and said second image comprises a plurality of secondelectrophoretic bands, where the vertical position of each of theplurality of first electrophoretic bands is associated with a predefinedmolecular weight such that the first image represents a vertical weightscale; a computer adapted for communicating with the imaging device, thecomputer having at least one processor to execute logic instructionsassociated with at least one computer software program and a memory forstoring the logic instructions, where the memory stores the first imageand the second image received from the imaging device, where at leastone computer software program contains instructions to create a layeredcomposite image from the first image and the second image, where thefirst image defines a first layer of the layered composite image and thesecond image defines a second layer of the layered composite image; andwhere the at least one computer software program contains instructionsto analyze the layered composite image, where the instructions foranalyzing comprise a first set of instructions and a second set ofinstructions, where the first set of instructions include performing amolecular weight determination by comparing information between thefirst image and the second image as it exists in the layered compositeimage, and where the second set of instructions include performing adensitometry determination by comparing information only within thesecond layer; and a user interface adapted to communicate at least onedetermination.
 19. The system of claim 18 where the computer furthercomprises at least one computer software program for noise reduction,adapted to reduce noise on a single layer.
 20. The system of claim 18where the user interface comprises a visual display for displaying thelayered composite image to a user.